Plasmalogen compositions and their use in neurodegenerative diseases treatment

ABSTRACT

The present invention is directed to plasmalogens and pharmaceutical compositions thereof which are useful for treating neurodegenerative diseases. Carbons 1 and 2 of the phosphoglyceryl backbone of the plasmaolgens have from 10-24 carbon atoms containing from 1 to 6 double bonds. The phosphoryl group is bonded to choline, ethanolamine, serine or inositol.

DESCRIPTION OF THE INVENTION

The present invention relates to the field of therapeutic chemistry andmore particularly to the one of phosphoglycerids.

More precisely, it consists in pharmaceutical compositions notablyintended for neurodegenerative diseases treatment, based onphosphoglyceroethers and phosphoglyceroesters.

STATE OF THE ART

Specifically, it consists in pharmaceutical compositions containingphosphoglyceroethers or plasmalogens, combined or not withphospholipids, admixed or diluted into inert, not toxic carriers,suitable for digestive or parenteral administration.

Phospholipids are defined as esters constituted of a glycerol moleculeesterified in position 1 and 2 with saturated or unsaturated fatty acidschains, and, in position 3 with a phosphoric acid derivative. Most ofnatural phospholipids contains saturated fatty acids chains such asstearic acid or palmitic acid. Moreover, the phosphoric group is boundto an aminoalkyl chain to form the phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol.These compounds which form the lecithins class are principally ofalimentary value.

The phospholipids, according to this invention, are characterized inthat the glycerol moiety is esterified and/or etherified by nonsaturated fatty chains.

Previous tests have shown the relevance of phospholipids containingpolyunsaturated fatty acids as an esterifying group.

Their function in nutrition and dietetic has already been the subject toseveral patent applications depending on whether they origin fromcerebral tissues or whether they come from the yolk of hens egg extractsthe food of which has been modified.

In fact, these phospholipids are carriers of fatty acids chains with ahigh number of carbons and they have a high unsaturation degree of then-3 series, especially the acid in C₂₂ :6.

The superiority in nutritional function of the cerebral phospholipids incomparison with other sources of n-3 fatty acids has been moreparticularly demonstrated (J. M. BOURRE J.Neur.Chem. 60 (1993)2018-2028).

SUMMARY OF THE INVENTION

The present invention thus relates to pharmaceutical compositions inwhich the phosphoglycerids are, partially or predominantly, substitutedby plasmalogens the formula of which is: ##STR1## wherein R is an alkylradical and, preferably, an alkenyl radical having one or several doublebonds up to 5 double bonds, and from 10 to 24 carbon atoms R' is alinear or branched, fatty, alkenyl of 10 to 24 carbon atoms and from 1to 6 double bonds and the nitrogen atom is inserted into a secondary,tertiary or quaternary, linear or cyclic structure.

The Merck Index (1989) page 1194, defines plasmalogens as aldehydogeniclipids extracted from the animal kingdom and nothing is known abouttheir therapeutic usefulness.

Their nomenclature is not already entirely determined and it is spokenas ethanolamine-phosphatidal or plasmalogen-ethanolamine, orplasmalogen-choline to define more precisely the nitrogenous structurecarried by the phosphoric group.

Phosphoglycero-ethers of the invention seem to have a high affinity forpolyunsaturated fatty acids with a long chain of the n-3 series and itcan be deduced therefrom a great physiological significance foreicosanoids. In this case, one characteristic of plasmalogen is that itcontains twice more from a polyunsaturated fatty acid (DHA i.e. C₂₂ : 6n-3) than other phospholipids.

Plasmalogens molecules are essentially present on the membrane and seemendowed with remarkable membrane exchange qualities. So, they would havea greatest metabolic activity at the level of the membranereconstruction.

Moreover, it has been shown that cultured mutant cells which do notsynthetize plasmalogen are very sensitive to agents generating freeradicals (O. MORAND J.Biol.Chem. 263 (1988) 11.797). This leads tosuppose a vitamin E-like protective effect of the plasmalogen with arole of anti-oxidizing in the oxidative stresses (B. ENGELMANNBiochem.Biophys.Res.Commun. 204 (1994) 1235).

The relevance of phosphoglycero-ethers appears during various geneticdiseases, as well in the animals as in the humans. So, among others,mutant mice breeds, used in laboratory, like "Jimpy" mice and "Quaking"mice have a depressed plasmalogens level, as well in the brain as in thespinal chord (M. H. HACK J.of Chromatogr. 145 (1978) 307), likewise pigssuffering from congenital trembling (D. S. PATTERSON J.Neurochem. 21(1973) 397).

Besides, in the humans, the Zellweger's cerebrohepatorenal syndrome is arare genetic disease with a brain degeneracy associated with anetherphospholipids insufficiency due to a lack of peroxisoms which aretheir biosynthesis sites.

In this disease, a great quantity of phospholipids esters with very longfatty acids chains of the n-6 serie is found in affected children'sbrain with, simultaneously, a very important decrease in the level ofthe n-3 series fatty acids, and particularly, of C₂₂ -6 (DHA) (A. POULOSBiochem.J. 253 (1988) 645).

Some other hereditary diseases induce neurological disorders ofdemyelinisation with a significant decrease in the level of plasmalogenat the cerebrospinal level, it is such the case of thePelizaeus-Merzbacher's leukodystrophic disease (J. M. BOURREEurop.J.Neurol. 17 (1978) 317), as well as in the Refsum infantiledisease too, the punctata chondrodysplasia and some others (A. K. HAJRAPlenum Press (1988) 369-380).

Some other degenerative diseases are associated with a perturbation ofthe phospholipids metabolism and, particularly, of plasmalogen at thecerebrospinal level, it is such the case in multiple sclerosis (J. M.BOGGS Neur.Chem.Res. 7 Aout 1982 p.953-954).

In several demyelinisation diseases and in ischemia, a rise of theplasmalogen level in cerebral tissues is observed, that is confirmingthe role of plasmalogens in myelin (L. A. HORROCK Adv.Exp.Med.Biol. 100(1978) 423-438).

The degradation of phospholipids membranes seems to be associated withAlzheimer's disease and a lowering of cerebral phosphatidylcholine andphosphatidylethanolamine level (R. M. NIBCH Proc.Natl.Acad.Sci.USA 89(1992) 1671), and also, a lowering of the rate of polyunsaturated fattyacids and, particularly C₂₂ : 6 (M. SODERBERZ Lipids 26 (1991)p.421-425) are reported.

Similar modifications of the same order have been reported in the Down'ssyndrome (B. W. BROOKSBANK Mal.Chem.Neuropathol. 11 (1989) p.157-185).

Preceeding informations have incited applicants to searchphospholipidethers sources and to measure their possible protectiveefficacy towards oxydizing agents or to degenerative processes.

Mammalian brain and spinal chord appear as the best sources ofphospholipidethers, in the form of plasmalogens; the used preparationcontains about 12% of plasmalogens with a high proportion ofpolyunsaturated n-3 fatty acids, the composition of which is following:

    ______________________________________                                        sphingomyeline                                                                            5 to 10%                                                          phosphatidylcholine                                                                       20 to 30% of which 1/20 is in plasmalogens form                   phosphatidylserine                                                                        15 to 20%                                                         phosphatidylinositol                                                                      3 to 5%                                                           phosphatidylethanol-                                                                      30 to 40% of which 2/3 are in plasmalogens                        amine       form                                                              phosphatidic acid                                                                         3 to 5%                                                           lysophospholipids                                                                         2 to 10% of which total plasmalogens are 10 to                                15%                                                               fatty acids n-3 total =                                                                   11%                                                               ratio n-6/n-3 =                                                                           15%                                                               ration C.sub.22 : 6 (n-3)/                                                                10%                                                               fatty acids total) =                                                          ______________________________________                                    

By comparison, hens eggs fed with a rich feeding of n-3 series fattyacids contain a lower proportion but however significative ofplasmalogens as well as a lesser quantity of n-3 serie long chain fattyacids as it is shown in Table I below:

                  TABLE 1                                                         ______________________________________                                        HEN EGGS PHOSPHOLIPIDS     ETHER LIPIDS                                       ______________________________________                                        Cholinephosphatidyl                                                                             70%      0                                                  Phosphatidyl ethanolamine                                                                       16%      2.8 to 4%                                          Other phospholipids                                                                             12%      0                                                  Total amount of lipids ethers                                                                            0.5 to 1%                                          n-3 fatty acids total                                                                           6 to 7%                                                     Ratio n-6/n-3     5 to 6%                                                     Ratio C.sub.22 : 6 (n-3)/                                                                       5%                                                          Fatty acids total                                                             ______________________________________                                    

This is the reason why a cerebral phospholipids preparation rich inplasmalogens (phosphoglycero-ethers) the composition of which and theplasmalogens content seems to be particularly suitable, is preferablyused.

In a more particular way, phospholipids used in the present inventioncontain from 5 to 25% of total plasmalogens and preferably from 10 to15%.

The compositions, according to this invention, also find a use to fightagainst intoxications or nervous degeneracies, diseases in which eithera deficiency in the biosynthesis or a phospholipids degradationcontaining an ester link or an ether link with an hydrocarbonatedpolyunsaturated fatty acid chain, particularly, the one of the n-3series, can be shown. It results in a more or less reversible damage ofthe nervous cell membrane wall.

The compositions according to this invention are intended to digestiveor parenteral administration. For these aims, there will be added toexcipients or diluents suitable for these ways of administration, suchas inert mineral products, for example, calcium carbonate, tricalciumphosphate, magnesium phosphate, alumina, colloidal silica, kaolin,clays, aluminium silicate, calcium silicate or iron oxide for thedigestive way of administration, of aqueous liquids for the parenteralway of administration.

The compositions according to this invention, can also contain organicinert supports of organic nature such as starches, dextrins, lactose,cellulose, cellulose synthetic derivatives, alginates, carrhagenates,fatty acids, waxes or resins.

The compositions according to this invention can also contain otheractive agents with complementary action such as the group B vitamins(vitamin B1, vitamin B2, vitamin B6, folic acid, pantothenic acid,pangamic acid, vitamin PP) or mineral salts or trace elements such asselenium, lithium or rubidium.

The compositions according to this invention thus appear in the form ofdrinkable vials, flasks, soft gelatine capsules, uncoated or coatedtablets, lozenges, granules or flavoured or not, sweetened or notpowders.

The compositions according to this invention can also appear in theliquid form such as, for example, a gelified preparation or a drinkablesuspension or even more an oil in water emulsion.

The present invention is also described with more details in thefollowing examples:

EXAMPLE I

A preparation based on phospholipids rich in ether lipids (plasmalogen)and preferably extracted from mammalians or fishes brains or spinalchord and optionally from hens eggs.

Cerebral phospholipids 10 to 300 mg of which 10 to 15% of ether lipids:

    ______________________________________                                        • Vitamin B1  0.30 to 4.2 mg                                            • Vitamin B2  0.30 to 4.8 mg                                            • Vitamin B6  0.35 to 6 mg                                              • Folic acid  30 to 600 μg                                           • Vitamin B12 0.15 to 3 μg                                           ______________________________________                                         For one capsule                                                          

EXAMPLE II

Capsules based on cerebral phospholipids rich in ether lipids(plasmalogen)

    ______________________________________                                        • Preparation according to example I                                                            70.0   g                                              • Lactose         175.5  g                                              • Vitamin E       20.0   g                                              • Microcrystalline cellulose                                                                    30.0   g                                              • Calcium stearate                                                                              4.5    g                                              ______________________________________                                         For 1000 capsules                                                        

EXAMPLE III

Capsules based on cerebral phospholipids rich in ether lipids(plasmalogen)

    ______________________________________                                        • Preparation according to example I                                                            65.0   g                                              • Magnesium oxide 75.0   g                                              • Zinc gluconate  100.0  g                                              • Maltodextrin    59.25  g                                              • Colloidal silica                                                                              0.75   g                                              ______________________________________                                         for 1000 capsules                                                        

EXAMPLE IV

Capsules based on cerebral phospholipids rich in ether lipids(plasmalogen)

    ______________________________________                                        • Preparation according to example I                                                            90.5   g                                              • Magnesium oxide 100.0  g                                              • Yeast autolysate titrated in vitamins                                                         55.0   g                                              of the B group                                                                • Carboxymethyl cellulose                                                                       53.6   g                                              • Colloidal silica                                                                              0.9    g                                              ______________________________________                                         for 1000 capsules                                                        

EXAMPLE V

Plasmalogen-based capsules

    ______________________________________                                        • Preparation according to example I                                                            100.0  g                                              • Lactose         770.0  g                                              • Magnesium stearate                                                                            20.0   g                                              • Colloidal silica                                                                              3.0    g                                              ______________________________________                                         for 1000 capsules                                                        

EXAMPLE VI

Tablets based on cerebral phospholipids rich in ether lipids(plasmalogen)

    ______________________________________                                        • Preparation according to example I                                                            95.5   g                                              • Magnesium oxide 250.0  g                                              • Sorbitol        637.5  g                                              • Microcrystalline cellulose                                                                    15.0   g                                              • Colloidal silica                                                                              2.5    g                                              ______________________________________                                         for 1000 capsules                                                        

EXAMPLE VII

Ampuls to be drunk based on cerebral phospholipids rich in ether lipids(plasmalogen)

    ______________________________________                                        • Preparation according to example I                                                            45.0   g                                              • Ammonium glycyrrhizinate                                                                      12.5   g                                              • Purified water  10     l                                              ______________________________________                                         for 1000 ampuls of 10 ml                                                 

EXAMPLE VIII

Sachets of drinkable powder based on cerebral phospholipids rich inether lipids (plasmalogen)

    ______________________________________                                        • Preparation according to example I                                                            100.0  g                                              • Magnesium oxide 500.0  g                                              • Zinc gluconate  125.0  g                                              • Vitamin E       30.0   g                                              • Maltodextrin with a high absorption                                                           2.5    g                                              capacity                                                                      • Diluent q.s.                                                          ______________________________________                                         for 1000 sachets 5 g                                                     

EXAMPLE IX

Injectible suspension based on cerebral phospholipids rich in etherlipids (plasmalogen)

    ______________________________________                                        • Cerebral phospholiplds                                                                  10 mg                                                       • Sodium chloride                                                                         15 mg                                                       • PPI water QS                                                          ______________________________________                                         for 1 ampuls of 5 ml                                                     

EXAMPLE X

Ether lipids dosage in mammalians brains extract has been performed bytwo methods:

A) Bidimensionnal chromatographic method

Firstly, total lipid extraction by the Folch method as it is describedby B. Entressangles, in "Manuel des corps gras" (editionsLavoisier-Paris) 1992-P. 1414-1418. Ether lipids are separated fromtotal phospholipids by bidimensionnal chromatography CIH vaporsintermediate hydrolysis. The thus hydrolysed ether lipids intocorresponding lyoderivatives, separate from diacylated phospholipidsduring the second dimension course.

B) By NMR spectrometry

Phospholipids are quantified by 31P! NMR spectroscopy using triphenylphosphate as an internal control. The samples are treated in a specialway to obtain lines and a good resolution. The resulting values are % ofphospholipids moles in relation to internal standard.

In order to convert % mole into % per weight, the values are multipliedby the corresponding phospholipid molecular weight.

Cholesterol and cerebrosides are quantitatively determined by 1H!NMRspectroscopy while by adding internal corresponding standards. Etherslipids data are obtained from signals included in the three spectra.Free fatty acids data are calculated from the 13C! NMR spectrum.Additionally to 31P! NMR measurements, 1H! NMR spectra and and a 13C!NMR spectrum have been carried out. Cholesterol and cerebrosides areadded as a standard in order to obtain quantitative values for these twolipids types through the 1H! NMR spectrum. By using the 13C! NMRspectrum, free acids and alkenyl ethers content, can be calculated.Table 2 shows the values obtained on cerebral phospholipids.

    ______________________________________                                        Total phospholipids                                                                             53.6%                                                       PC-Ether                  0.7%                                                PE-Ether                  12.0%                                               Cholesterol       7.0%                                                        Cerebrosides      9.0%                                                        Free fatty acids  5.0%                                                        Total             74.6%                                                       ______________________________________                                         PC-Ether = phosphatidylcholine ether                                          PEEther = phosphatidylethanolamine ether                                 

EXAMPLE XI

Lipids ethers preparations from pig cerebral tissues

Cerebral phospholipids according to this invention are obtained byextracting pig brains removed from freshly slaughtered animals accordingto the following methods:

pig brains are removed from freshly slaughtered animals stemming frombreedings without any infectious diseases and rigorously followed byveterinary services for the sanitary conditions.

brains are immediately frozen at -20° C. and kept at this temperature

brains are then reduced to a temperature comprised between -5° to 0° C.before getting through an industrial mincer and grinders, in order toobtain a liquid paste the content of water of which, is about 80%.

the brain paste is transferred to the top of an atomizing room whereinwater is immediately evaporated through a hot air flow at 190°/195° C.

the resulting powder is fed into a reactor which contains a mixture ofaliphatic hydrocarbons based on hexane and kept under stirring.

after filtration, the liquid phase is vacuum-concentrated and give riseto a crude extract

the crude extract is then run into acetone in the presence of analimentary antioxidising agent

the obtained precipitate is filtered under nitrogen pressure

the collected product is vacuum-dried and contains the purified cerebralphospholipids.

EXAMPLE XII

Pharmacological effect demonstration of the preparations according tothis invention.

A plasmalogen-rich (phosphoglycero ethers) cerebral phospholipidspreparation has been used as a protective agent in a certain number ofanimal tests.

a) On pyrithiamin-induced experimental neuritis

Pyrithiamin is an antimetabolite which at a 5 mg/Kg IP dose, induces inthe mice a tetanic hyperpolarization.

Phospholipids protective activity was tested, on the one hand as apreventive treatment i.e. administered at the same time than pyrithiaminand, on the other hand, as a curative agent, that is when the firstneurological symptoms appear.

Results are reported Table 3 below.

It is stated that the protective activity during preventive treatment istotal until doses as low as 2.5 mg of phospholipids per 10 g of food.Knowing that a 20 g mouse eats about 3 g of food a day, the effectivedose is about 0.35 mg of phospholipids rich in plasmalogens according tothe invention, per animal.

                  TABLE 3                                                         ______________________________________                                        Dosis of phospholipids in                                                                    Preventive effect                                                                          Curative effect                                   10 g food in the groups under                                                                Con-             Con-                                          experiment     trols   Experim. trols Experim.                                ______________________________________                                        10 mg          +++     0        +++   +                                       5 mg           +++     0        +++   ++                                      2,5 mg         +++     0        +++   ++                                      2 mg           +++     +        +++   ++                                      1 mg           +++     +++      +++   +++                                     ______________________________________                                         +++Acute polyneuritis: al animals die in few days                             ++Attenuated symptoms: survival of more than 50% of animals                   +light symptoms: all animals survive                                          0 no symptom                                                             

b) Protective effect of phospholipids rich in plasmalogen onoxotremorine-induced neurological symptoms

Oxotremorine induces in the mice very characteristic static tremblings,of the Parkinson's disease type, as well as peripheral symptoms such aslachrymation and salivation.

                  TABLE 4                                                         ______________________________________                                        Dosis of phospholipids                                                                    Duration of  Intensity of the symptoms                            in 10 g food                                                                              the treatment                                                                              as a function of time after                          in the groups under                                                                       with phospholipids                                                                         arrest of the treatment                              experiment  before tremorine                                                                           1 j    2 j  3 j  4 j                                 ______________________________________                                         0 mg                    +++    +++                                           50 mg       3 days       0      0    ++                                       30 mg       3 days       0      0    ++                                       20 mg       3 days       0      ++   +++                                      10 mg       3 days       0      +++                                            0 mg                    +++    +++                                           50 mg       1 days       0      0    +++                                      30 mg       1 days       0      0    +++                                      20 mg       1 days       0      +++                                           10 mg       1 days       0      +++                                           ______________________________________                                    

Table 4 clearly shows that 50 mg of phospholipids per 10 g of foodadministered during 3 days before oxotremorine injection, completelyinhibit symptoms appearance (tremblings), this inhibition to theoxotremorine action lasts 3 days.

c) Effects of phospholipids rich in plasmalogens on experimentalallergic encephalomyelitis

An encephalomyelitis is induced among 20 rats by injecting a spinalchord suspension (Freund's adjuvant). Disease grows up from the 10th tothe 14th day. These animals divided into 3 groups have been treatedevery day with increasing dosis of phospholipids.

                  TABLE 5                                                         ______________________________________                                        Dosis of brain                                                                           Lack of symptoms                                                   phospholipids I.M                                                                        after 7 j                                                                              after 14 j                                                                             after 21 j                                                                            Not cured                                ______________________________________                                        0,01 mg    1        2        1       16                                       0,02 mg    8        17               3                                         0,5 mg    6        18               2                                        ______________________________________                                    

The curative effect is thus very clear.

EXAMPLE XIII

Clinical effect demonstration of preparations according to thisinvention

A clinical trial have been undertaken with a group of children sufferingfrom severe mental handicaps. One treated group of 104 children has beencompared to an untreated group of 85 children aged from 1 to 14.Treatment consists in a 30 mg phospholipids supply a day per os for 6 to12 months periods as the case may be. Psychometric tests, I.Q.measurements or electroencephalographic recording have been achievedaccording to the age or the symptomatology.

It will be noted in Table 6 below, that more of six children out of ten,have gained an important benefit from this treatment.

                                      TABLE 6                                     __________________________________________________________________________           RESULTS TREATED CHILDREN                                                                            RESULTS UNTREATED CHILDREN                              Number                                                                            None                                                                             Weak                                                                              Significant                                                                        Very marked                                                                         Number                                                                            None                                                                              Weak                                                                              Significant                                                                        Very marked                     __________________________________________________________________________    DOWN'S  40 0  13  21    6    24   6  15  3    0                               syndrom                                                                       LITTLE'S                                                                              22 6   6   8    2    11   9   2  0    0                               disease                                                                       Post meningo-                                                                 encephalitic                                                                          7  0   3   4    0     7   5   2  0    0                               condition                                                                     Post    9  0   3   4    2     5   4   1  0    0                               ictero-nuclear                                                                condition                                                                     Endogenous                                                                            13 2   2   6    3    18  11   7  0    0                               debility                                                                      Petit mal                                                                             4  0   0   3    1     6   3   2  1    0                               Grand mal                                                                             9  1   2   4    2    10   6   3  1    0                               TOTAL  104 9  29  50   16    81  44  32  5    0                                          8,6%                                                                             27,8%                                                                             48%  15,3%     42,3%                                                                             30;7%                                                                             4,8%                                                   63,3%                                                       __________________________________________________________________________

I claim:
 1. A composition for treating neurodegenerative diseasescomprising an anti-neurodegeneratively effective amount of glyceratedphospholipids from mammalian brains or fish containing 5 to 25% byweight of a compound ##STR2## wherein R is alkenyl of 10 to 24 carbonatoms containing 1 to 5 double bonds, R' is alkenyl of 10 to 24 carbonatoms containing 1 to 6 double bonds and B is selected from the groupconsisting of choline, ethanolamine, serine and inositol and an inertpharmaceutical carrier.
 2. The composition of claim 1 containing 10 to15% by weight of the compounds.
 3. The composition of claim 1 furthercontaining at least one member selected from the group consisting ofvitamins B₁, B₂ and B₆, vitamin PP and folic acid.
 4. The compound ofclaim 1 further containing at least one magnesium salt.
 5. Thecomposition of claim 1 further containing at least one trace elementselected from the group consisting of selenium, lithium and rubidium. 6.The method of treating neurodegenerative diseases in humansadministering to humans in need thereof an antineurodegenerativelyeffective amount of glycerated phospholipids from mammalian brains orfish containing 5 to 25% by weight of a compound of Formula ##STR3##wherein R is alkenyl of 10 to 24 carbon atoms containing 1 to 5 doublebonds, R' is alkenyl of 10 to 24 carbon atoms containing 1 to 6 doublebonds and B is selected from the group consisting of choline,ethanolamine, serine and inositol.
 7. The method of claim 6 wherein theglycerated phospholipids also contain at least one member selected fromthe group consisting of vitamins B₁, B₂ and B₆, vitamin PP and folicacid.